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WO2013019975A1 - Compositions et procédés de traitement du cancer - Google Patents

Compositions et procédés de traitement du cancer Download PDF

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Publication number
WO2013019975A1
WO2013019975A1 PCT/US2012/049375 US2012049375W WO2013019975A1 WO 2013019975 A1 WO2013019975 A1 WO 2013019975A1 US 2012049375 W US2012049375 W US 2012049375W WO 2013019975 A1 WO2013019975 A1 WO 2013019975A1
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Prior art keywords
cancer
cells
dtpp
bso
aur
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PCT/US2012/049375
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English (en)
Inventor
Douglas R. SPITZ
Michael K. SCHULTZ
Kyle KLOEPPING
Yueming Zhu
Nukhet AYKIN-BURNS
Max S. Wicha
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University Of Iowa Research Foundation
University Of Michigan
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Priority to US14/236,879 priority Critical patent/US9801922B2/en
Publication of WO2013019975A1 publication Critical patent/WO2013019975A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
    • A61K31/341—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide not condensed with another ring, e.g. ranitidine, furosemide, bufetolol, muscarine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/662—Phosphorus acids or esters thereof having P—C bonds, e.g. foscarnet, trichlorfon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/66—Phosphorus compounds
    • A61K31/675—Phosphorus compounds having nitrogen as a ring hetero atom, e.g. pyridoxal phosphate
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7004—Monosaccharides having only carbon, hydrogen and oxygen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca

Definitions

  • the present invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising an XTPP agent that increases reactive oxygen species (ROS) levels in cancer cell mitochondria, an inhibitor of hydroperoxide metabolism, and a pharmaceutically acceptable diluent or carrier.
  • ROS reactive oxygen species
  • Examples of reactive oxygen species include superoxide and hydrogen peroxide (i.e., 0 2 * ⁇ , H 2 0 2 ).
  • the XTPP agent comprises a triphenylphosphonium (TPP) molecule or a pharmaceutically acceptable salt thereof.
  • TPP triphenylphosphonium
  • triphenylphosphonium is any molecule containing a triphenylphosphine cation ( + PPh 3 ) moiety.
  • the XTPP agent is + PPh 3 -X-R Y " ;
  • X is a (C 2 -C 50 )alkyl
  • R is H, N 3 , triazole optionally substituted with one or more (e.g. 1 or 2) (C 4 -C 8 )alkyl or quinone optionally substituted one or more (e.g. 1, 2 or 3) (C C 6 )alkyl or -0(C 1 -C 6 )alkyl; and
  • Y " is a counterion
  • alkyl is defined as a straight or branched hydrocarbon.
  • an alkyl group can have 2 to 50 carbon atoms (i.e, (C 2 -C5 0 )alkyl), 1 to 10 carbon atoms (i.e., (C 1 -C 1 o)alkyl), 1 to 8 carbon atoms (i.e., (CrC 8 )alkyl) or 1 to 6 carbon atoms (i.e., (Ci-C alkyl).
  • alkyl groups include, but are not limited to, methyl (Me, - CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1 -propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1 -butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl-l -propyl (i-Bu, i-butyl, - CH 2 CH(CH 3 ) 2 ), 2-butyl (s-Bu, s-butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (n
  • counterion is a pharmaceutically acceptable counterion such as a pharmaceutically acceptable anion (e.g. CI “ , Br “ , ⁇ , CH 3 S0 3 " , CF3SO3 " or /?-CH 3 C 6 H 4 S0 3 ).
  • a pharmaceutically acceptable anion e.g. CI “ , Br “ , ⁇ , CH 3 S0 3 " , CF3SO3 " or /?-CH 3 C 6 H 4 S0 3 ).
  • R imparts hydrophilicity or reactive properties cytotoxic to cancer cells.
  • X is -(CH 2 ) 10 -.
  • the XTPP agent that increases reactive oxygen species (ROS) levels in cancer cell mitochondria is:
  • the pharmaceutical composition includes an inhibitor of glutathione synthesis or hydroperoxide metabolism comprising L-buthionine-[S,R]- sulfoximine (BSO), (S-triemylphosphinegold(I)-2,3,4,6-tetra-0-acetyl-l -thio- -D- glucopyranoside Auranofin (AUR), or a combination of BSO and AUR.
  • BSO L-buthionine-[S,R]- sulfoximine
  • AUR S-triemylphosphinegold(I)-2,3,4,6-tetra-0-acetyl-l -thio- -D- glucopyranoside Auranofin
  • Other compounds that could also be used for this purpose include inhibitors of catalase (i.e.
  • 3-aminotriazole inhibitors of glucose metabolism (i.e., bromopyruvate and 2-deoxyglucose), inhibitors of peroxiredoxins, inhibitors of glutathione peroxidases, inhibitors of dehydrogenase enzymes that regenerate NADPH, inhibitors of thioredoxin reductase, inhibitors of glutathione reductase, inhibitors of glutathione transferases, and inhibitors of transcription factors as well as signal transduction proteins that regulate thiol mediated hydroperoxide metabolism (i.e., Nrf-2, AP-1, NFkB, AKT, ERKl/2, p38, EGFR, and IGFR).
  • compositions including DTPP with inhibitors of hydroperoxide metabolism would include feeding patients diets high in respiratory directed substrates including ketogenic diets, Atkins style diets, and pharmacological doses of IV vitamin C which would be expected to further enhance the differential metabolic production of pro-oxidants in cancer vs. normal tissues.
  • the present invention provides a method for treating cancer in a mammal, comprising administering a composition described above to the mammal.
  • the agent that increases reactive oxygen species (ROS) levels in cancer cell mitochondria also called “an XTPP agent” or “XTPP” herein
  • ROS reactive oxygen species
  • the present invention provides a method for inducing clonogenic cell killing and cellular apoptosis of a cancerous cell, comprising contacting the cancerous cell with an effective clonogenic cell killing or apoptosis-inducing amount of the composition described above.
  • the XTPP agent and inhibitor of hydroperoxide metabolism or glutathione synthesis are administered sequentially rather than in a single composition.
  • the present invention provides a method for increasing the anticancer effects of a conventional cancer therapy (i.e., radio- and/or chemo-therapy) on cancerous cells in a mammal, comprising contacting the cancerous cell with an effective amount of the composition described above and administering an additional conventional cancer therapy modality.
  • a conventional cancer therapy i.e., radio- and/or chemo-therapy
  • the additional cancer therapy is chemotherapy and/or radiation.
  • the XTPP and inhibitor of hydroperoxide metabolism or glutathione synthesis are administered sequentially rather than in a single composition.
  • the composition does not significantly inhibit viability of comparable non-cancerous cells.
  • the present invention provides a method for selectively inducing oxidative stress in a cancer cell in a mammal in need of such treatment comprising administering to the mammal an effective amount of the composition described above.
  • the XTPP and inhibitor of hydroperoxide metabolism are administered sequentially rather than in a single composition.
  • the mammal is a human.
  • the cancer is breast cancer, prostate cancer, lung cancer, pancreas cancer, head and neck cancer, ovarian cancer, brain cancer, colon cancer, hepatic cancer, skin cancer, leukemia, melanoma, endometrial cancer, neuroendocrine tumors, carcinoids, neuroblastoma, tumors arising from the neural crest, lymphoma, myeloma, or other malignancies characterized by aberrant mitochondrial hydroperoxide metabolism.
  • the cancer is the above cancers that are not curable or not responsive to other therapies.
  • the cancers are hormone dependent or hormone-independent epithelial cancers.
  • the tumor is reduced in volume by at least 10%. In certain embodiments, the tumor is reduced by any amount between 1-100%. In certain embodiments, the tumor uptake of molecular imaging agents, such as fluorine- 18 deoxyglucose, fluorine- 18 thymidine or other suitable molecular imaging agent, is reduced by any amount between 1-100%. In certain embodiments the imaging agent is fluorine- 18 deoxyglucose, fluorine- 18 thymidine or other suitable molecular imaging agent. In certain embodiments, the mammal's symptoms (such as flushing, nausea, fever, or other maladies associated with cancerous disease) are alleviated.
  • the mammal's symptoms such as flushing, nausea, fever, or other maladies associated with cancerous disease
  • the composition is administered intraveneously, orally, subcutaneously, or as an aerosol. In certain embodiments of the methods described above, the composition is administered intraveneously at a dosage of 5-200 micromols/kg/day of XTPP, such as 20-130 micromols/kg/day of XTPP . In certain embodiments of the methods described above, the composition is administered orally at a dosage of 5-200 micromols/kg/day of XTPP, such as 20-130 micromols/kg/day of XTPP.
  • the present invention provides a method for treating cancer in a subject, comprising administering to the subject an effective amount of XTPP and an inhibitor or inhibitors of hydroperoxide metabolism and/or an inhibitor of glutathione metabolism so as to treat the cancer.
  • the present invention provides a composition comprising a decyl-triphenylphosphonium (DTPP) or a pharmaceutically acceptable salt thereof, and an inhibitor of hydroperoxide metabolism for use in the treatment of cancer, wherein the composition is to be administered to a patient that has cancer or is at risk for developing cancer.
  • DTPP decyl-triphenylphosphonium
  • the present invention provides a composition comprising a decyl-triphenylphosphonium (DTPP) or a pharmaceutically acceptable salt thereof, and an inhibitor of hydroperoxide metabolism for use in inducing cellular apoptosis of a cancerous cell, wherein the composition is to be administered to a patient that has cancer or is at risk for developing cancer.
  • DTPP decyl-triphenylphosphonium
  • Aldehyde dehydrogenase activity positive (ALDH+) cancer cells also known as early progenitor cancer stem cells
  • BSO, AUR inhibitors of glutathione and thioredoxin metabolism
  • Table 2 Measurement of AUR, DTPP and NAC interactions with Ellman's reagent. Different concentrations (0.01 mM-10 mM) of NAC, DTPP and AUR solutions were prepared. Equal moles of NAC, DTPP or AUR from each concentration were added together along with DTNB [5,5' - dithio-bis- ( 2-nitrobenzoic acid)], respectively. For each combination, the same moles of single agents were also added with DTNB. DMSO was added into NAC alone solution to maintain the same DMSO level in NAC+DTPP/AUR combinations. The absorbance of DTNB's reduction to 2-nitro-5-thiobenzoate (TNB) was then measured spectrophotometrically at 412 nM.
  • TNB 2-nitro-5-thiobenzoate
  • NAC and AUR interaction test sample tubes were incubated in a 4%, 37°C incubator for at least lh.
  • NAC and DTPP interaction test sample tubes were incubated in a 4%, 37°C incubator for 24h. The results show that both NAC and AUR were capable of reducing DTNB confirming their reactivity with disulfide containing compounds like DTNB.
  • Panel ID shows in vivo treatment with Au, BSO and DTPP alone and in combinations results in a decrease of ALDH + cells in Sum 159 xenografts.
  • Mice growing Sum 159 xenograft tumors were treated with 100 ⁇ DTPP in drinking water for 2 weeks followed by i.p. injections of BSO 675 mg/kg followed in 2 hrs with Au 2.7 mg/kg. The day following injections tumors were harvested, digested and stained for ALDH positive cells. Each bar represents an average of at least three tumors. Error bars are SEM. * p ⁇ 0.05 vs. control.
  • Figures 3A-3B Clonogenic survival of SUM159 and MDA-MB231 cells treated with BSO, DTPP and AUR in the presence of 20 mM NAC.
  • Asynchronously growing cultures of SUM 159 (panel A) and MDA-MB231 (panel B) were plated as described in Figure 1. After 48 hours, cells were given fresh complete HMECs media and treated with 100 ⁇ BSO +/ 1 ⁇ DTPP in the presence or absence of 20 mM NAC for 24 hours. 500 nM AUR were added 15mins before the trypsinization. cells were then collected and plated for clonogenic survival.
  • FIGS 4A-4C MitoSOX oxidation in SUM159, MDA-MB231 and HMECs cells treated with BSO, DTPP and AUR
  • SUM159 Panel A
  • MDA-MB231 Panel B
  • HMECs Panel C
  • Monolayer cultures were harvested and trypsinized, washed once with PBS and labeled 20 minutes with 2 ⁇ MitoSOX (in 0.1% DMSO) in PBS containing 5 mM pyruvate at 37°C. Each sample was then analyzed for the Mean fluorescence Intensity (MFI) of 10,000 cells by flow cytometry.
  • MFI Mean fluorescence Intensity
  • Figures 5A-5D CDCF3 ⁇ 4 oxidation sensitive and CDCF oxidation insensitive probe labeling of BSO, DTPP, and AUR exposed SUM159 and HMECs cells.
  • PEGSOD+/PEGCAT 100 U/mL each for 24 hours. Control received PEG alone (18 ⁇ ) for 24 hours. 500 nM AUR were added 15mins before the trypsinization. Cells were then collected and plated for clonogenic survival.
  • SUM159 BSO, DTPP and AUR treated SUM159.
  • Asynchronously growing cultures of SUM159 were plated and treated as described in Figure 1. Cells were harvested and scraped in PBS at 4°C. Whole cell homogenates were used for native gel redox western blot analysis of thioredoxin reductase activity. The results show that these drug combinations induce oxidative stress in the cancer cells.
  • FIG 8 Intracellular thioredoxin reductase (TRR) activity measured in BSO, DTPP, and AUR exposed SUM159 cells.
  • TRR thioredoxin reductase
  • Asynchronously growing cultures of SUM159 were plated and treated as described in Figure 1.
  • Cells were harvested and scraped in PBS at 4°C.
  • FIG. 9 Catalase inhibitable CDCF3 ⁇ 4 oxidation in BSO, DTPP, and AUR exposed SUM159 cells.
  • Asynchronously growing cultures of SUM159 were plated and treated as described in Figure 1.
  • 100 U/ml PEG-CAT or 18 ⁇ PEG alone were given 2 hours before and during CDCFH 2 labeling to cells.
  • Monolayer cultures were harvested and trypsinized, washed once with PBS then labeled in PBS with either CDCFH 2 (10 ⁇ g/mL, in 0.1% DMSO 15 minutes) at 37°C.
  • Figure 10 DHE oxidation of BSO,DTPP and AUR exposed SUM159 cells.
  • MFI Mean fluorescence Intensity
  • FIG. 11 TPP variants synthesized to examine the effect molecular chain substituents on cancer cell specific cytotoxicity.
  • Figure 12 Azido-decylTPP and Bis-TPP MB231 clonogenic assay. These data show that increasing concentrations of azido-decylTPP have a dose dependent cytotoxic effect on breast cancer cells, while the bis-decylTPP compound does not in human cancer cells.
  • MB231 cells were treated with 0.5 ⁇ , 1.0 ⁇ , and 2.0 ⁇ azido-decylTPP and bis-TPP and incubated for 24 hrs.
  • DMSO was added to control dishes to a final concentration of 0.1%.
  • FIG. 13 Azido-decylTPP and Bis-TPP Hec50co clonogenic assay. These data show that increasing concentrations of azido-decylTPP have a dose dependent cytotoxic effect on human endometrial cancer cells, while the bis-decylTPP compound has a more modest effect in Hec50co cells. Hec50co human endometrial cancer cells were treated with 0.5 ⁇ , 1.0 ⁇ , and 2.0 ⁇ azido-decylTPP and bis-TPP and incubated for 24 hrs. DMSO was added to control dishes to a final concentration of 0.1 %. Following a 24 hr.
  • Figures 14A-14B MTT survival fraction analysis of A375 melanoma cells looking at the effect of variation in TPP molecular chain substituent length in the presence and absence of BSO with comparison to standard of care dacarbazine:
  • pentyl-TPP has little cytotoxicity in the presence of BSO up to 2 ⁇ concentration, while TPP conjugates with longer tails 10, 15, 20 atoms have significant cytotoxicity in the presence of BSO;
  • B The cytotoxicity of a TPP conjugate with a 20 carbon chain length in the tail function has significant cytotoxicity in the absence of BSO, while the effect is lessened for shorter tail conjugates.
  • Figure 15 DTPP treated mitochondria electron transport chain activity assays looking at the specific mitochondrial electron transport chain complexes that TPP based compounds inhibits. The activity of electron transport chain complexes I-IV was measured
  • DTPP a lipophilic cation that localizes to cancer cell mitochondria
  • inhibitors of hydroperoxide metabolism i.e., L-bulMonine-S,R-sulfoximine, BSO ( ⁇ ), Auranofin (S- triethylphosphinegold(I)-2,3 ,4,6-tetra-O-acetyl- 1 -thio-b-Dglucopyranoside), AUR (500nM)] to treat breast cancer cells in vitro.
  • BSO+DTPP treatment could induce at least additive (and possibly greater than additive) clonogenic cell killing in MDA- MB231 and SUM 159 human breast cancer cells, that was significantly less toxicity than was seen in normal human mammary epithelial cells.
  • AUR 500nM
  • BSO ⁇ DTPP could further sensitize cancer cells to the cytotoxicity of BSO ⁇ DTPP.
  • AURDH aldehyde dehydrogenase
  • increases in parameters indicative of oxidative stress including steady-state levels of CDCF3 ⁇ 4, and MitoSOX oxidation, were also observed in BSO, DTPP and AUR treated human breast cancer cells, relative to normal cells.
  • N- acetylcysteine, a non-specific thiol antioxidant, and PEG-SOD and PEG-CAT could rescue toxicity of BSO, DTPP and AUR exposed SUM159 and MDA-MB231 cells.
  • Triphenylphosphonium (TPP) salts can be reacted with alcohols, alkyl halides, and carboxylic acids, which allow them to be used as starting materials for the synthesis of a large variety of chemical derivatives, e.g., XTPP agents.
  • Charged molecules generally cannot pass through cell membranes without the assistance of transporter proteins because of the large activation energies need to remove of associated water molecules.
  • the charge is distributed across the large lipophilic portion of the phosphonium ion, which significantly lowers this energy requirement, and allows the TPP to pass through lipid membranes.
  • the phosphonium salts accumulate in mitochondria due to the relatively highly negative potential inside the mitochondrial matrix.
  • compositions of the present invention utilize XTPP agents that have activity in treating cancer cells, in that the XTPP agents preferentially localize to cancer cells, as compared to the comparable normal cells because cancer cells are often characterized by abnormal mitochondrial oxidative metabolism (Aykin- Burns N, Ahmad IM, Zhu Y, Oberley LW, and Spitz DR: Increased levels of superoxide and hydrogen peroxide mediate the differential susceptibility of cancer cells vs. normal cells to glucose deprivation. Biochem. J. 2009; 418:29-37. PMID: 189376440) and altered mitochondrial membrane potential (Chen LB: Mitochondrial membrane potential in living cells, Ann. Rev. Cell Biol. 1988; 4:155-81), relative to normal cells.
  • the XTPP agent comprises a triphenylphosphonium (TPP) molecule or a pharmaceutically acceptable salt thereof.
  • TPP triphenylphosphonium
  • triphenylphosphonium is any molecule containing a triphenylphosphine cation ( + PPh 3 ) moiety.
  • the XTPP agent is + PPh 3 -X-R;
  • X is a (C 2 -C 50 )alkyl
  • R is H, N 3 , triazole optionally substituted with one or more (e.g. 1 or 2) (C 4 -C 8 )alkyl or quinone optionally substituted one or more (e.g. 1, 2 or 3) (C 1 -C 6 )alkyl or -0(C 1 -C 6 )alkyl; and
  • alkyl is defined as a straight or branched hydrocarbon.
  • an alkyl group can have 2 to 50 carbon atoms (i.e, (C 2 -Cs 0 )alkyl), 1 to 10 carbon atoms (i.e., (Ci-C 10 )alkyl), 1 to 8 carbon atoms (i.e., (CrC ⁇ alkyl) or 1 to 6 carbon atoms (i.e., (Ci-C 6 alkyl).
  • alkyl groups include, but are not limited to, methyl (Me, - CH 3 ), ethyl (Et, -CH 2 CH 3 ), 1 -propyl (n-Pr, n-propyl, -CH 2 CH 2 CH 3 ), 2-propyl (i-Pr, i-propyl, -CH(CH 3 ) 2 ), 1 -butyl (n-Bu, n-butyl, -CH 2 CH 2 CH 2 CH 3 ), 2-methyl- 1 -propyl (i-Bu, i-butyl, - CH 2 CH(CH 3 ) 2 ), 2-butyl (s-Bu, s-butyl, -CH(CH 3 )CH 2 CH 3 ), 2-methyl-2-propyl (t-Bu, t-butyl, -C(CH 3 ) 3 ), 1-pentyl (n-pentyl, -CH 2 CH 2 CH 2 CH 3 ), 2-pentyl (n
  • R imparts hydrophilicity or reactive properties cytotoxic to cancer cells.
  • X is -(CH 2 )!o-.
  • the XTPP agent that increases reactive oxygen species (ROS) levels in cancer cell mitochondria is;
  • the inventors discovered that the addition of inhibitors of hydroperoxide metabolism ia glutathione and/or thioredoxin dependent pathways to a composition including an XTPP agent, that selectively enhances clonogenic cell killing via oxidative stress and accumulation of oxidative damage to critical biomolecules (i.e., proteins, lipids, and nucleic acids), in human cancer cells, relative to normal human cells.
  • This selective property of the drug combination(s) for clonogenically inactivating cancer cells is the result of inherent differences in pro-oxidant levels generated in cancer vs. normal cells as by products of oxidative and reductive metabolism necessary for maintenance of cell viability and reproduction.
  • cancer cells demonstrate increased levels of reactive oxygen species (i.e., superoxide, hydroperoxides, and reactive species derived from the oxidation of proteins, lipids, and nucleic acids) due to fundamental differences in cancer vs. normal cell metabolism of oxygen.
  • reactive oxygen species i.e., superoxide, hydroperoxides, and reactive species derived from the oxidation of proteins, lipids, and nucleic acids
  • the addition of these inhibitors of hydroperoxide metabolism to a composition including XTPP also enhances the efficacy of conventional radiation and chemotherapies used to treat human cancers.
  • the inhibitors of hydroperoxide metabolism are L-buthionine-[S,R]-sulfoximine (BSO), (S- triethylphosphinegold(I)-2,3 ,4,6-tetra-O-acetyl- 1 -thio-b-Dglucopyranoside Auranofin (AUR), or a combination of BSO and AUR.
  • BSO and AUR or a combination of these two compounds are employed to inhibit thiol mediated hydroperoxide metabolism by both glutathione- and thioredoxin-dependent pathways which causes oxidative stress and accumulation of oxidative damage to critical biomolecules (i.e., proteins, lipids, and nucleic acids) in cancer versus normal cells resulting in cancer cell specific clonogenic cell killing in both early progenitor cancer stem cells as well as all other cancer cells capable of continued mitotic activity.
  • critical biomolecules i.e., proteins, lipids, and nucleic acids
  • inhibitors of catalase i.e., 3- aminotriazole
  • inhibitors of glucose metabolism i.e., bromopyruvate and 2-deoxyglucose
  • inhibitors of peroxiredoxins inhibitors of glutathione peroxidases
  • inhibitors of glutathione peroxidases inhibitors of glutathione
  • dehydrogenase enzymes that regenerate NADPH, inhibitors of thioredoxin reductase, inhibitors of glutathione reductase, inhibitors of glutathione transferases, and inhibitors of transcription factors as well as signal transduction proteins that regulate thiol mediated hydroperoxide metabolism (i.e., Nrf-2, AP-1, NFkB, AKT, ERK1/2, p38, EGFR, and IGFR).
  • compositions including XTPP with inhibitors of hydroperoxide metabolism would include feeding patients diets high in respiratory directed substrates including ketogenic diets, Atkins style diets, and pharmacological doses of IV vitamin C which would be expected to further enhance the differential production of pro- oxidants mentioned previously in cancer vs. normal tissues.
  • Compositions to Kill Cancer Cells via Oxidative Stress would include feeding patients diets high in respiratory directed substrates including ketogenic diets, Atkins style diets, and pharmacological doses of IV vitamin C which would be expected to further enhance the differential production of pro- oxidants mentioned previously in cancer vs. normal tissues.
  • the present invention provides compositions to kill cancer cells via oxidative stress.
  • XTPP and inhibitors of hydroperoxide metabolism are combined into a single composition.
  • the two components are administered individually or sequentially.
  • the effective amount of the XTPP and the inhibitors of hydroperoxide metabolism does not significantly affect the viability of comparable normal cells.
  • the effective amount causes the killing of less than 100% (e.g., less than about 95%, less than about 90%, less than about 85%, less than about 80%, less than about 75%, less than about 70%, less than about 65%, less than about 60%, less than about 55%, less than about 50%, less than about 45%, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, or less than about 5%) of the comparable normal cells.
  • the composition could kill breast cancer cells present in a mammal, but kill fewer than 100% of the normal breast cells, e.g., only 5% of the normal breast cells.
  • beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment can also mean prolonging survival as compared to expected survival if not receiving treatment.
  • Those in need of treatment include those already with the condition or disorder as well as those prone to have the condition or disorder or those in which the condition or disorder is to be prevented.
  • the XTPP and inhibitors of hydroperoxide metabolism may be administered by any route appropriate to the condition to be treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, intradermal, intrathecal and epidural), transdermal, rectal, nasal, topical (including buccal and sublingual), vaginal, intraperitoneal, intrapulmonary and intranasal.
  • the dosage of the XTPP and inhibitors of hydroperoxide metabolism will vary depending on age, weight, and condition of the subject. Treatment may be initiated with small dosages containing less than optimal doses, and increased until a desired, or even an optimal effect under the circumstances, is reached.
  • the dosage is about 1 ⁇ g/kg up to about 100 ⁇ g/kg body weight, e.g., about 2 ⁇ g/kg to about ⁇ g/kg body weight of the subject, e.g., about 8 ⁇ g/kg to about 35 ⁇ g/kg body weight of the subject.
  • Higher or lower doses are also contemplated and are, therefore, within the confines of this invention.
  • a medical practitioner may prescribe a small dose and observe the effect on the subject's symptoms. Thereafter, he/she may increase the dose if suitable.
  • the XTPP and inhibitors of hydroperoxide metabolism are administered at a concentration that will afford effective results without causing any unduly harmful or deleterious side effects, and may be administered either as a single unit dose, or if desired in convenient subunits administered at suitable times.
  • a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration.
  • the therapeutip agent may be introduced directly into the cancer of interest via direct injection.
  • routes of administration include oral, parenteral, e.g., intravenous, slow infusion, intradermal, subcutaneous, oral (e.g., ingestion or inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Such compositions typically comprise the XTPP and inhibitors of hydroperoxide metabolism and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration.
  • solvents dispersion media, coatings, antibacterial and anti-fungal agents, isotonic and absorption delaying agents, and the like.
  • the use of such media and agents for pharmaceutically active substances is well known in the art.
  • Solutions or suspensions can include the following components: a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)), fixed oils, a polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), glycerine, or other synthetic solvents; antibacterial and antifungal agents such as parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose.
  • a sterile diluent such as water for injection, saline solution (e.g., phosphate buffered saline (PBS)),
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • Prolonged administration of the injectable compositions can be brought about by including an agent that delays absorption.
  • agents include, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • parenteral preparation can be enclosed in ampules, disposable syringes, or multiple dose vials made of glass or plastic.
  • Dosage unit form refers to physically discrete units suited as unitary dosages for an individual to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the dosage unit forms of the invention are dependent upon the amount of a compound necessary to produce the desired effect(s).
  • the amount of a compound necessary can be formulated in a single dose, or can be formulated in multiple dosage units. Treatment may require a one-time dose, or may require repeated doses.
  • cancer cells may increase glucose metabolism as a compensatory mechanism to protect against intracellular ROS. If this is the case, then inhibiting glucose or hydroperoxide metabolism while forcing cells to derive energy from respiration should preferentially kill cancer cells, relative to normal cells.
  • BSO is the one that has been best characterized in animal model studies and human clinical trials.
  • BSO is a Glutathione (GSH) synthesis inhibitor. It can inhibit glutamate cysteine ligase (GCL) activity, therefore inhibiting GSH synthesis and decreasing the GSH level.
  • GSH and GSH dependent enzymes play a very important role in hydroperoxides metabolism, and therefore decreasing GSH level by BSO could significantly increase the oxidative stress in cancer cells and increase their susceptibility compared to normal cells. Nonetheless, the implementation of BSO as single anticancer agent in vivo has been a disappointment.
  • Mitochondria might be a highly promising, relatively undervalued anti-cancer target.
  • Research by the inventors demonstrated that mitochondrial produced ROS significantly contribute to the differential susceptibility of cancer and normal cells to glucose deprivation- induced cytotoxicity and oxidative stress (Aykin-Burns N, Ahmad IM, Zhu Y, Oberley L, and Spitz DR. Increased levels of superoxide and hydrogen peroxide mediate the differential susceptibility of cancer cells vs. normal cells to glucose deprivation. Biochem J. 2009;
  • DTPP Decyl- Triphenylphosphonium
  • CSC cancer stem cell
  • Bis-TPP was synthesized by refluxing triphenylphosphine (0.8 g, 2.0 mMol) with a 10- fold excess of 1, 10-dibromodecane (1.0 g, 20.0 mMol) in 10 mL benzene for 3 days at 80 °C.
  • the final product was purified by silica gel chromatography using the following solvents: (50% hexanes / 50% ethyl acetate; 100% ethyl acetate; 10% methanol / 90% ethyl acetate; 50% methanol / 50% ethyl acetate; and 100% methanol).
  • Azido-decylTPP was synthesized by refluxing triphenylphosphine (0.5 g, 2.0 mMol) (AlphaAesar®, L02502, Ward Hill, Ma) with a 25-fold excess of 1, 10-dibromodecane (10.5 g, 50.0 mMol) (AlphaAesar ® , L07383, Ward Hill, Ma) in 10 mL benzene for 24 hrs. at 80 °C yielding a (lO-bromodecyl)triphenylphosphonium intermediate.
  • the intermediate (0.77 g, 1.6 mMol) was refluxed with a 5- fold excess of NaN 3 (0.52 g, 8.0 mMol) in a 30 mL 1 : 1 mixture of EtOH and water for 24 hrs. at 80 °C yielding (lO-azidodecyl)triphenylphosphonium.
  • the final product was purified by silica gel chromatography using the following solvents: (50% hexanes / 50% ethyl acetate; 100% ethyl acetate; 10% methanol / 90% ethyl acetate; 50% methanol / 50% ethyl acetate; and 100% methanol).
  • MDA-MB231 and SUM159 human breast cancer cells were obtained from the American Type Culture Collection (Manassas, VA, USA). MDA- MB231 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS; Hyclone, Logan, UT, USA). SUM159 cells were maintained in Ham's F-12 medium (Gibco Invitrogen, Carlsbad, CA, America) containing lOmM HEPES (Gibco Invitrogen, Carlsbad, CA, USA), 10 ng/ml insulin (Sigma, St. Louis, MO), lmg/ml hydrocortisone (Sigma, St.
  • HMEC human mammary epithelial cells
  • MEBM serum free medium LiM serum free medium
  • rhEGF human epidermal growth factor
  • BPE bovine pituitary extract
  • rhEGF human epidermal growth factor
  • BPE bovine pituitary extract
  • All the cell cultures were maintained in humidified 37°C incubator with 5% C0 2 and 4% 0 2 . All experiments were done using exponentially growing cell cultures at 50-70 % confluence. DMSO and PBS were used as the vehicle control in all experiments.
  • BSO, NAC, Polyethylene glycol (PEG), polyethylene glycol-catalase (PEG-CAT), and polyethylene glycol-superoxide dismutase (PEG-SOD) were obtained from Sigma Chemical Co. (St. Louis, MO, USA).
  • Auranofin (AUR) was obtained from ICN Biochemicals (Aurora, OH).
  • Decyl-triphenylphosphonium (DTPP) was obtained from Dr. Michael P. Murphy at Medical Research Council Mitochondrial Biology Unit, Cambridge CB2 0XY, UK. All drugs were used without further purification.
  • Drugs were added to cells at the final concentrations of 100 ⁇ BSO, 1 ⁇ DTPP, 500 nM AUR, 20mM NAC, 18 ⁇ PEG, 100 U/ml PEG-CAT and 100 U/ml PEG-SOD.
  • Stock solutions of 0.01 M BSO, 5 U/ ⁇ PEG-CAT and 5 U/ ⁇ PEG-SOD were dissolved in PBS and the required volume was added directly to the cells to achieve the desired final concentration (PEG alone at the same concentration (18 ⁇ ) was added as the control).
  • Stock solutions of 1 mM TPP and ImM AUR were dissolved in dimethyl sulfoxide (DMSO), respectively, with the final
  • Clonogenic cell survival To determine whether the DTPP exposure alters the cell proliferation in MDA-MB231, SUM159 and HMECs, 150,000 cells/dish of MDA-MB231 and SUM 159 cells, and 300,000 cells/dish HMECs were plated in 60 mm tissue culture dishes. After 48 hours, at 24 hours prior to each clonogenic survival experiment, media in all the dishes were changed to HMECs media and then treated with 100 ⁇ BSO and 1 ⁇ DTPP for 24h. Cells were treated with AUR for 15 minutes prior to each clonogenic survival experiment.
  • Cieciura S. J. Action of x-rays on mammalian cells. II. Survival curves of cells from normal human tissues. J Exp Med 106:485-500; 1957; Spitz, D. R.; Malcolm, R. R.; Roberts, R. J. Cytotoxicity and metabolism of 4-hydroxy-2-nonenal and 2-nonenal in H202 -resistant cell lines. Do aldehydic by-products of lipid peroxidation contribute to oxidative stress? Biochem J 267:453-459; 1990).
  • the ALDEFLUOR kit (StemCell Technologies, Durham, NC, USA) was used to determine the subpopulation of cancer cells with a high ALDH enzymatic activity. SUM 159 human breast cancer cells were plated and treated as described earlier in clonogenic survival experiment. After the exposure, the cells were trypsinized and washed with PBS once then re-suspended in ALDEFLUOR assay buffer containing ALDH substrate (BAAA, 1 ⁇ / ⁇ per 1x106 cells) and incubated for 40 minutes at 37°C.
  • ALDH substrate BAAA, 1 ⁇ / ⁇ per 1x106 cells
  • each sample of cells received 4 ⁇ g/ml of Hoechst 33258 (Molecular Probes,
  • DHE Dihvdroethidium
  • MitoSOXTMred oxidation to estimate mitochondrial superoxide production To determine if DTPP can alter steady-state levels of superoxide originating from mitochondria in SUM 159, MDA-MB231 and HMEC cells, the cationic superoxide sensitive dye,
  • MitoSOXTMred (Molecular Probes), was used. Cells were plated and treated as described earlier in clonogenic survival experiment. After the exposure, cells were trypsinized and washed with 5 mM/L pyruvate containing PBS once then labeled with MitoSOXTMred (2 ⁇ , in 0.1% DMSO, 20 minutes) at 37°C. After labeling, cells were kept on ice. Samples were analyzed using a FACScan flowcytometer (Becton Dickinson Immunocytometry System, INC., Mountain View, CA) (excitation 488 nm, emission 585 nm band-pass filter). The mean fluorescence intensity of 10,000 cells was analyzed in each sample and corrected for autofluorescence from unlabeled cells.
  • FACScan flowcytometer Becton Dickinson Immunocytometry System, INC., Mountain View, CA
  • Glutathione analysis SUM159 Cells were plated and treated with BSO, DTPP and AUR as described above. When cells were grown to 70-80% confluency on 100 mm dishes and scraped in PBS at 4°C, centrifuged, and the cell pellets were frozen at -20°C until analysis. Samples were thawed and whole homogenates were prepared as described and total glutathione (GSH +GSSG) was determined using a recycling method (Spitz, D. R.; Malcolm, R. R.; Roberts, R. J. Cytotoxicity and metabolism of 4-hydroxy-2-nonenal and 2-nonenal in H202-resistant cell lines. Do aldehydic by-products of lipid peroxidation contribute to oxidative stress? Biochem J 267:453-459; 1990; Griffith, O. W. Determination of glutathione and glutathione disulfide using glutathione reductase and 2-vinylpyridine. Anal Biochem
  • Bicinchoninic Acid Protein Assay was performed using the BCATM Protein Assay Kit from Pierce Biotechnology (Rockford, IL). The assay was performed according to manufacturer's instructions, using the "Enhanced Protocol.”
  • Thioredoxin Reductase activity was measured by using Thioredoxin Reductase Assay Kit purchased from Sigma (St Louis, MO). 400,000 cells/ 100mm dishes of SUM159 cells were plated and treated with BSO, DTPP and AUR as described above. When cells were grown to 70-80% confluency on 100 mm dishes and scraped in PBS at 4°C, centrifuged, and the cell pellets were frozen at -20°C until analysis. The assay was performed according to manufacturer's instructions.
  • the difference between the sample rate with and without the TrxPv inhibitor was taken to be the difference caused by TrxR activity in the sample.
  • Each sample was then normalized to protein, by using the Lowry protein assay, as described previously (Lowry, O. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275; 1951).
  • the thioredoxin reductase activity of each sample was then normalized as a percentage of that of the control cells.
  • Thioredoxin Redox Western The human thioredoxin- 1 protein level was determined by Thioredoxin redox western. Approximately 2-3 million treated or untreated SUM159 cells were lysed in G-lysis buffer (50 mM Tris-HCl, pH 8.3, 3 mM EDTA, 6 M guanidine-HCl, 0.5% Triton X-100) containing 50 mM iodoacetic acid (pH 8.3). The lysate was incubated in the dark for thirty minutes. The lysates were then centrifuged in G-25 microspin columns (GE Healthcare). Protein was then quantified, from the eluent by performing a Bradford protein assay, as previously described (Bradford, M. M.
  • the primary antibody was then removed, the blot was washed in PBST for 10 minutes three times, with constant shaking, before being incubated for 1 h with the secondary antibody (Rabbit anti-goat IgG, HRP labeled (Santa Cruz Biotechnology). The blot was then washed again for 10 minutes three times in PBST before being treated with HRP
  • chemiluminescence detection reagents Renaissance, NEN
  • the protein was then visualized by exposing the blot to X-ray film for 2-5 min in a dark room with a film cassette, before developing the film.
  • Measure of AUR, DTPP and NAC interaction by Ellman's reagent To determine if NAC could direct react with AUR or DTPP, different concentrations of NAC, DTPP and AUR solutions were prepared.
  • a NAC concentration standard curve were fist constructed by adding different concentrations of NAC with DTNB and measuring spectrophotometrically the absorbance of DTNB 's reduction to 2-nitro-5-thiobenzoate (TNB) at 412 nM.
  • NAC, DTPP or AUR were added together along with DTNB, respectively.
  • same moles of single agents were also added with DTNB.
  • DMSO was added into NAC alone solution to maintain the same DMSO level in NAC+DTPP/AUR combinations.
  • the absorbance of DTNB's reduction to 2-nitro-5- thiobenzoate (TNB) was then measured spectrophotometrically at 412 nM.
  • NAC and AUR interaction test sample tubes were incubated in a 4%, 37°C incubator for at least lh.
  • NAC and DTPP interation test sample tubes were incubated in a 4%, 37°C incubator for 24h.
  • TPP derivatives preferentially accumulate within cancer cell mitochondria, decrease the mitochondrial membrane potential, decrease oxygen consumption, and lead to an increase in free radical species. This data suggests that TPP derivatives act as inhibitors of the electron transport chain (ETC). To determine the specific mechanisms of action that TPP derivatives have on the ETC at the molecular level, we aimed to determine if
  • decyltriphenylphosphonium inhibits one or more specific complex of the ETC.
  • Spectrophotometric assays were performed on isolated mitochondria to evaluate how DTPP affects the activity of each ETC complex. Results show that DTPP significantly reduced the activity of complexes I and III, while negligible affects were observed in complexes II and TV, thus supporting our preliminary data that TPP derivatives inhibit oxidative phosphorylation.
  • Liver mitochondria harvested for ETC assays Livers were harvested from mice and placed in cold homogenizing medium (0.25 M sucrose, 5 mM hepes, 0.1 mM EDTA, 0.1% fatty acid free bovine serum albumin (BSA), (pH 7.25)). Samples were homogenized on ice using a glass dounce homogenizer and centrifuged at 1000 x g for 10 min at 4° C.
  • cold homogenizing medium 0.25 M sucrose, 5 mM hepes, 0.1 mM EDTA, 0.1% fatty acid free bovine serum albumin (BSA), (pH 7.25). Samples were homogenized on ice using a glass dounce homogenizer and centrifuged at 1000 x g for 10 min at 4° C.
  • decyltriphenylphosphonium 500 uM to simulate the concentration of the compound in active, respirating mitochondria; however, final DTPP concentration in all complex activity assays was approximately 10 uM following sample dilution after the initial incubation.
  • Total protein content was determined by Bradford assay (Biorad) and all electron transport chain enzyme activities were normalized to the total protein content.
  • Mitochondria (resuspended in 20 mM potassium phosphate buffer, (pH 7.0)) were lysed due to freeze thawing and divided into four samples.
  • Sample one contained complex I working buffer (25 mM potassium phosphate buffer (pH 7.2), 5 mM magnesium chloride, 2 mM potassium cyanide, 2.5 mg/mL BSA, 0.13 mM NADH), antimycin A (200 ⁇ g/mL), coenzyme Ql (7.5 mM), and mitochondria (0.37 ⁇ g/ ⁇ L).
  • Sample two contained complex I working buffer, antimycin A, coenzyme Q 1 , rotenone (200 ⁇ g/mL), and mitochondria (0.37 ⁇ g/ ⁇ L).
  • Sample three contained complex I working buffer, antimycin A, coenzyme Ql, DTPP, and mitochondria (0.36 ⁇ g/ ⁇ L).
  • Sample 4 contained complex I working buffer, antimycin A, coenzyme Ql, rotenone, DTPP, and mitochondria (0.36 ⁇ / ⁇ ). Samples were mixed and incubated for 1 min at 30° C.
  • the Complex II activity assay measured the rate of absorbance change due to the reduction of 2,6-dichloroindophenol (DCIP) ( ⁇ -19.1 mM "1 cm “1 ) by coenzyme Q in the presence and absence of succinate.
  • Mitochondria (resuspended in 20 mM potassium phosphate buffer, (pH 7.0)) were lysed due to freeze thawing and divided into four samples.
  • Sample one contained complex II working buffer (25 mM potassium phosphate buffer (pH 7.2), 5 mM magnesium chloride, 2 mM potassium cyanide, 2.5 mg/mL BSA), 25 mM potassium phosphate buffer, and mitochondria (0.37 ⁇ g/ ⁇ L).
  • Sample two contained complex II working buffer, 0.2 M succinate, and mitochondria (0.37 ⁇ g/ ⁇ L).
  • Sample three contained complex II working buffer, 25 mM potassium phosphate buffer, DTPP, and mitochondria (0.36 ⁇ g/ ⁇ L).
  • Sample four contained complex II working buffer, succinate, DTPP, and mitochondria (0.36 ⁇ g/ ⁇ uL). Samples were mixed and incubated for 10 min at 30° C.
  • Coenzyme Q2 was reduced by adding 1 N HC1 and potassium borohydride to 35 mM coenzyme Q2 until the reaction mixture turned from bright yellow to clear. The clear solution was transferred to a new tube and HC1 was added to keep coenzyme Q2 reduced.
  • Fresh mitochondria (resuspended in 20 mM potassium phosphate buffer, (pH 7.0)) were divided into four samples.
  • Sample one contained complex III working buffer (25 mM potassium phosphate buffer (pH 7.2), 5 mM magnesium chloride, 2 mM potassium cyanide, 2.5 mg/ml BSA, 0.5 mM n-dodecyl ⁇ - maltoside), rotenone (200 ⁇ g/mL), 1.5 mM cytochrome c, and 3.5 mM coenzyme Q2.
  • Sample two contained complex III working buffer, rotenone, cytochrome c, coenzyme Q2, and mitochondria (2.97 ⁇ ⁇ ).
  • Sample three contained complex III working buffer, rotenone, cytochrome c, coenzyme Q2, and DTPP.
  • Sample one contained complex IV working buffer (20 mM potassium phosphate buffer (pH 7.0), 0.5 mM n-dodecyl ⁇ -maltoside), 1.5 mM reduced cytochrome c, and mitochondria (0.37 ug/uL).
  • Sample two contained complex IV working buffer, reduced cytochrome c, DTPP, and mitochondria (0.36 ug/uL).
  • results in Table 1 represent the percentage of ALDH positive cell of 100,000 cell events in each treatment group. From the table, it was noted that the average ALDH positive cells population in SUM 159 was around 2.61%. It is noted that BSO as a single agent alone was not able to decrease the ALDH positive cell population. In contrast, with AUR or DTPP treatment, the percentage of ALDH positive cell population decreased from 2.61% to 1.11% or 1.16%, respectively.
  • BSO+AUR, BSO+DTPP, BSO+DTPP+AUR combinations further decreased ALDH positive cells from 2.61 % to 0.45%, 0.64%, and 0.17%, respectively.
  • an in vivo nude mouse experiment with SUM 159 human breast cancer cells was performed.
  • the results of this study showed that Aur+BSO+DTPP was also able to deplete ALDH positive cancer stem cells from human cancer cell populations in vivo grown as xenografts in nude mice ( Figure ID).
  • NAC was also demonstrated to be able to fully protect the SUM159 and MDA-MB231 cells from BSO+AUR induced toxicity and significantly rescue SUM159 and MDA-MB231 cells from clonogenic killing induced by BSO+DTPP and BSO+AUR+DTPP drug treatments.
  • Ellman's regent test for interaction was accomplished. Ellman's reagent (5,5'-dithiobis-(2-nitrobenzoic acid) or DTNB) is a chemical used to quantify the number or concentration of thiol groups in a sample.
  • NAC 2-nitro-5- thiobenzoate
  • NTB 2 2-nitro-5- thiobenzoate
  • Table 2 clearly shows that the 412 nm absorbance of the NAC+DTPP group with 24 hr incubation was same as that of NAC alone, suggesting there is no direct chemical interaction between NAC and DTPP during the drug treatment.
  • the 412 nm absorbance of NAC+AUR is the sum of the absorbance of NAC and AUR alone, which indicates that AUR was able to reduce DTNB. This might be due to the gold cation, which can facilitate cleavage of the S-S bond of the disulfide in DTNB and further reduce to NTB 2 " . This could have some significance for how AUR is able to inhibit thioredoxin reductase activity but the mechanistic details of the reaction should be further explored. Because absorbance of the combination group equals the sum of two single drugs, the data in table 2 also shows that there is no direct reaction between NAC and AUR that is capable of inactivating AUR. Given all these facts, these results support the conclusion that metabolic oxidative stress plays a causal role in BSO+DTPP+AUR-mediated breast cancer cell killing in a thiol dependent fashion that this toxicity is inhibited by a thiol antioxidant .
  • DTPP could significantly enhance the clonogenic cell killing mediated by BSO and AUR and this toxicity can be protected by NAC.
  • This result suggested that DTPP might exhibit its biological effects by increasing oxidative stress and thiol oxidation in SUM 159 and MDA-MB231 cells.
  • ROS reactive oxygen species
  • DTPP is a mitochondrial targeted cationic lipophilic molecule, it is possible that DTPP could interfere with the mitochondrial electron transport chain leading to more one electron reductions of 02 to form superoxide and hydrogen peroxide which could further react with oxygen to generate superoxide.
  • CDCFH 2 crosses cellular membranes and is enzymatically hydrolysed by "intracellular” esterases, and then can be further oxidized to produce a green fluorescent compound (CDCF) trapped inside of the cell for detection by flow cytometry.
  • DTPP was shown to significantly increase the CDCFH 2 oxidation (indicative of increased prooxidant production) in SUM 159 cells. Since GSH or Trx are important small molecule thiol antioxidants necessary for detoxifying hydroperoxides and other species capable of oxidizing critical thiol residues in proteins, we wanted to know if DTPP induced could increase steady-state level of ROS that could be synergistically enhanced with BSO and AUR to maximize their toxicity towards cancer cells. Therefore, levels of total GSH and GSSG were measured in drug treated SUM159 cells to determine if disruptions in glutathione metabolism were involved in the oxidative stress and toxicity caused by BSO, AUR, DTPP.
  • BSO+AUR could significantly increase the %GSSG from 1.13% to 4.88% and 10.81%, respectively.
  • AUR as a single agent was not capable of altering the glutathione metabolism compared to control.
  • DTPP could modestly affect glutathione metabolism.
  • DTPP is a mitochondrial targeted molecule, and DTPP did induce increased steady-state levels of ROS from mitochondria. Therefore, a mitochondrial GSH analysis should also be accomplished to fully understand if DTPP could selectively affect mitochondrial GSH metabolism.
  • thioredoxin reductase Another key component of thioredoxin metabolism is thioredoxin reductase, which was suggested to be able to exhibit pro-survival signaling and help cancer cells to escape oxidative stress induced cytotoxicity.
  • SUM159 cells treated with BSO, AUR, DTPP as previously described data in Figure 8 showed that 15 min AUR exposure could significantly inhibit TRR activity, decreasing from 135 mU/mg protein to 10 mU/mg protein.
  • DTPP could increase the TrxR activity while still causing significant cytotoxic effects. It is possible that this TRR activity increase might act as an adaptive response against the cytotoxicity caused by DTPP induced-oxidative stress. Alternatively the TRR activity increase could be due to an adaptive response to elevated oxidized Thioredoxin by DTPP exposure. Since we did not see a significantly increase in oxidized hTrx-1, again, this data again suggests that more experiments (i.e., hTrx-2 western blot) should be accomplished to fully understand the mechanisms and consequences of DTPP induced TRR activity.
  • cancer cells are under persistent oxidative stress because of increased steady-state levels of superoxide and hydroperoxides, relative to normal cells. Moreover, it has been also clearly documented that cancer cells have altered antioxidant levels (i.e., SOD, CAT, GSH). In order to cope with these excessive ROS, cancer cells have been hypothesized to increase glycolysis and pentose phosphate cycle activity to compensate for excess hydroperoxide production by direct deacetylation of hydroperoxides by pyruvate (formed during glycolysis), as well as regeneration of NADPH in the pentose cycle, which could provide electrons for glutathione and thioredoxin dependent peroxidase systems.
  • antioxidant levels i.e., SOD, CAT, GSH
  • Glutathione is one of the most important small molecule antioxidants in the detoxification of hydroperoxides; decreased glutathione levels would induce incapability of hydroperoxides removal.
  • GSH also played an important role in tumor drug resistance. Therefore, inhibitors of hydroperoxides metabolism, by depleting GSH levels, have been tested in cancer therapy.
  • BSO is a relatively specific inhibitor of gamma- glutamylcysteine synthetase, the rate-limiting step in GSH synthesis.
  • BSO as a single agent in cancer therapy was not very effective in some clinical trials possibly because of redundancy of in peroxide metabolic pathways. It is also known that anti-cancer treatment developments that focus on agents that target a single molecule or signaling pathway are unlikely to meet the expectation due to the versatile nature of tumor cells. Thus, adding other toxic agents with BSO treatment should be considered.
  • the excessive ROS production in cancer cells compared to normal cells might provide conditions for selectively targeting the cancer cells with agents that can increase the ROS production and result in oxidative stress mediated cell death in cancer cells, while normal cells should possess enough antioxidants to deal with the extra ROS production. Therefore, an agent that can increase the metabolic ROS production combined with an inhibitor of hydroperoxide production should be anticipated to further preferentially kill cancer cells, relative to normal cells. Additionally, recent research showed that mitochondrial electron transport chain blockers (ETCB) like Antimycin A (Ant A) or rotenone (Rot) could increase steady-state levels of 0 2 * ⁇ , and H 2 0 2 ; cause the accumulation of glutathione disulfide; and enhance 2DG-induced cell killing.
  • ETCB mitochondrial electron transport chain blockers
  • mitochondria may be the major source of pro-oxidant production during the 2DG exposure. It is therefore logical to predict that inhibiting glucose and hydroperoxide metabolism while increasing the mitochondrial ROS production could contribute to the excess oxidative stress in cancer cells, and this increase of oxidative stress could preferentially kill cancer cells relative to normal cells.
  • DTPP is a lipophilic cation, which can pass directly through phospholipid bilayers due to its large hydrophobic surface area lowering the activation energy for uptake, and which accumulates further into
  • mitochondrial accumulation of lipophilic cations could increase the permeability of the mitochondrial inner membrane and inhibit mitochondrial enzymes nonspecifically, affecting mitochondrial ROS production, inhibiting respiration, and leading to cell death.
  • lipophilic cations could be used as anti-cancer drugs because many cancer cells have a higher mitochondrial membrane potential than normal cells, which could lead to selective uptake. It has also been shown that lipophilic cations can disrupt cell function and selectively kill the cancer cells in vivo and in vitro. However, there is relatively little knowledge about the mechanism of how DTPP induces mitochondrial dysfunction and ROS production.
  • the TPP + group is thought to associate with the phospholipid head groups at the mitochondrial inner membrane matrix surface, while the decyl chain could insert to the bilayer, it is possible that the inserted chain could affect the electron transport chain in mitochondria and increase the probability of one electron reductions of 0 2 to form superoxide.
  • the selective accumulation of DTPP in cancer cell mitochondria might inhibit mitochondrial respiration, a major pathway to provide reducing equivalents.
  • damage to the mitochondria can cause changes in mitochondrial permeability and the release of apoptotic factors that could further contribute to the DTPP induced cell death in human breast cancers, relative to normal cells. Therefore, we hypothesized that DTPP might affect mitochondrial function by increasing mitochondrial ROS and further contribute to the toxicity of inhibitors of hydroperoxide metabolism.
  • Trx thioredoxin reductase
  • Trx thioredoxin reductase
  • Trx thioredoxin reductase
  • Trx thioredoxin
  • TrxR initiates a pro-survival signaling cascade in response to ROS induced cytotoxicity.
  • TRR/Trx could also contribute to many other cell functions including DNA synthesis, gene transcription, cell growth and transformation, and resistance to cytotoxic agents that induce oxidative stress and apoptosis.
  • AUR could significantly sensitize MDA-MB231 and SUM 159 to the BSO or BSO+DTPP combination but without affecting the proliferation of HMECs.
  • BSO+AUR and BSO+AUR+DTPP treatments still induced selective cell killing in SUM 159 and MDA-MB231 cells compared to HMECs ( Figure 1C).
  • AUR thioredoxin reductase inhibitor
  • CSC cancer stem cell
  • results in table 1 showed BSO alone did not decrease the population of ALDH positive cells whereas AUR alone or DTPP alone could decrease this population to around 50%. Moreover, when BSO was combined with DTPP or AUR+DTPP, these combinations even more significantly decreased the ALDH positive cell population. Although further determination of whether these signals were truly representative of the CSC population is needed, this result suggested BSO, DTPP and AUR exposure could deplete the CSC population. And these results also indicate targeting the intrinsic ROS difference between cancer stem cells and normal cells might be an effective approach in cancer therapy.
  • Trx-1 and TrxR assay 15 min AUR was able to significantly decrease TrxR activity and induce a 2-fold increase of the ratio of oxidized Trx-1 to reduced Trx-1 ( Figure 7 and 8).
  • DTPP was not able to change the ratio of oxidized to reduced Trx-1 , but could induce a 2-fold increase of TrxR activity ( Figure 7 and 8). It is unclear why changes were caused by DTPP exposure.
  • glutathione assay we only measured the whole cell GSH level and it is possible as afore-mentioned that the primary site of ROS production is mitochondria. Thus it is possible a mitochondrial GSH assay will be better way to investigate the role of DTPP on glutathione metabolism.
  • TrxR activity increase could be caused by cancer cell up-regulation of TRR activity to act against the DTPP caused cytotoxicity.
  • elevated mitochondrial ROS production after DTPP exposure could lead to an increased ratio of oxidized to reduced Trx-2. This increase of Trx-2 oxidation might further activate TRR to more efficiently recycle the oxidized Trx-2.
  • TPP based drugs were carried out on isolated mitochondria to determine the specific ETC protein complex interactions with TPP based drugs that drive superoxide production. These assays demonstrated that TPP based drugs interact with and inhibit complexes I and III, while little interaction was observed with complexes II and IV in the mitochondrial ETC.
  • CDCFH 2 5-(and-6)-carboxy-2 ' ,7 ' -dichlorodihydrofluorescein diacetate

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Abstract

L'invention concerne des compositions et des procédés de traitement du cancer grâce à un agent qui augmente les niveaux des dérivés actifs de l'oxygène (DAO) dans les mitochondries de cellules cancéreuses ("un agent xTPP" - tétraphénylporphyrines) ou un sel pharmaceutiquement acceptable de celui-ci, un inhibiteur du métabolisme des hydroperoxydes et un diluant ou un support pharmaceutiquement acceptable.
PCT/US2012/049375 2011-08-03 2012-08-02 Compositions et procédés de traitement du cancer WO2013019975A1 (fr)

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US11098073B2 (en) 2017-04-20 2021-08-24 Oxford University Innovation Limited Triphenylphosphonium-tethered tetracyclines for use in treating cancer
US11161862B2 (en) 2017-04-20 2021-11-02 Oxford University Innovation Limited Phosphonium-ion tethered tetracycline drugs for treatment of cancer
WO2018193114A1 (fr) * 2017-04-20 2018-10-25 Novintum Biotechnology Gmbh Tétracyclines attachées au triphénylphosphonium destinées à être utilisées dans le traitement du cancer
WO2018193113A1 (fr) * 2017-04-20 2018-10-25 Novintum Biotechnology Gmbh Médicaments de tétracycline attachés à un ion phosphonium pour le traitement du cancer
US12006553B2 (en) 2017-05-19 2024-06-11 Lunella Biotech, Inc. Companion diagnostics for mitochondrial inhibitors
US11738034B2 (en) 2017-11-24 2023-08-29 Lunella Biotech, Inc. Triphenylphosphonium-derivative compounds for eradicating cancer stem cells
WO2020214754A1 (fr) * 2019-04-16 2020-10-22 Lunella Biotech, Inc. Composés alkyl-tpp pour le ciblage des mitochondries et traitements anticancéreux
EP3955935A4 (fr) * 2019-04-16 2023-01-18 Lunella Biotech, Inc. Composés alkyl-tpp pour le ciblage des mitochondries et traitements anticancéreux
JP7586835B2 (ja) 2019-04-16 2024-11-19 ルネラ・バイオテック・インコーポレーテッド ミトコンドリア標的化及び抗癌治療のためのアルキルｔｐｐ化合物
WO2021238761A1 (fr) * 2020-05-27 2021-12-02 Beijing Bjut Science And Technology Park Co., Ltd Composition thérapeutique et procédé de traitement d'une infection à coronavirus
WO2022026210A1 (fr) 2020-07-31 2022-02-03 University Of Iowa Research Foundation Compositions pour le traitement d'un trouble hyperprolifératif avec un inhibiteur de la synthèse du gsh et une composition anticancéreuse
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WO2023091689A1 (fr) 2021-11-19 2023-05-25 University Of Iowa Research Foundation Utilisation associée de radiothérapie dirigée par mcr1 et d'inhibition du point de contrôle immunitaire dans le traitement d'un mélanome
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